*New Insights about the Influence of Yeasts Autolysis on Sparkling Wines Composition and Quality DOI: http://dx.doi.org/10.5772/intechopen.101314*

#### **Figure 3.**

*Changes in the polysaccharide content of sparkling wines over ageing time. LMW, low molecular weight fraction (40–7.5 kDa); IMW, intermediate molecular weight fraction (180–40 kDa); HMW, high molecular weight fraction (>180 kDa). Adapted from Pons-Mercadé et al. [15].*

#### **Figure 4.**

*Changes in the protein content of sparkling wines over ageing time. LMW, low molecular weight fraction (50–25 kDa); IMW, intermediate molecular weight fraction (75–50 kDa); HMW, high molecular weight fraction (>75 kDa). Adapted from Pons-Mercadé et al. [15].*

approach consisting of analyzing the wine model solutions that had been in contact with the lees for a year. **Figure 5** shows the obtained results. The polysaccharides released in this model wine solution by the lees (**Figure 5A**) increased between the first (roughly 4 mg/L) and the second (roughly 6 mg/L) year of aging while decreasing progressively in the later vintages, reaching a minimum value in the ninth year of ageing (roughly 0.90 mg/L). The mannose concentration obtained from the hydrolysis of this polysaccharide (**Figure 5B**) showed very similar, which would confirm that they were mainly mannoproteins.

The total protein concentration released from the lees of different aging times in a model wine (**Figure 5C**) showed a similar pattern to that of the polysaccharides reaching a maximal value in the third year (roughly 0.32 mg/L) and a minimum value in the ninth year of aging (roughly 0.17 mg/L).

To reproduce the cumulative release effect of polysaccharides and proteins over the lees aging time, the concentrations of both macromolecules released from the first to the ninth year were added (**Figure 5D** and **E**). This simple approach shows a clear increase in the accumulated concentrations of both macromolecules over the ageing time. The total accumulation of polysaccharides at the end of the 9 years was 26.6 mg/L, while that of proteins was 2.4 mg/L. These values should be taken with caution since they reflect just one approach. Nevertheless, this data indicates that

*New Insights about the Influence of Yeasts Autolysis on Sparkling Wines Composition and Quality DOI: http://dx.doi.org/10.5772/intechopen.101314*

#### **Figure 5.**

*Changes in the polysaccharide and protein content of sparkling wines over ageing time. A. Total polysaccharides; B. Mannose; C: Total protein; D. Polysaccharides released by lees; E: Proteins released by lees. Adapted from Pons-Mercadé et al. [15].*

the release of polysaccharides and proteins from the lees during the ageing process was much lower than the usual concentrations present in these sparkling wines.

Another approach was carried out to illustrate the distribution of polysaccharides and proteins according to their origin (from the autolysis of lees or base wine) throughout the aging time. **Figure 6** shows the percentage of polysaccharides (A) and proteins (B) from lees autolysis or base wines for total concentration in the sparkling wines. This figure clearly shows that the percentage of polysaccharides and proteins from lees autolysis was extremely low in the young sparkling wines. That means that during the first year of ageing, sparkling wine had only 2% of proteins and 3% of polysaccharides from the lees. These percentages increased as

#### **Figure 6.**

*Distribution of proteins and polysaccharides of sparkling wine according to their origin. Adapted from Pons-Mercadé et al. [15].*

the ageing time increased and reached maximal values in the seventh year of ageing (14% for polysaccharides and 16% for proteins).

It is necessary to point out that only 88% of sparkling wines from AOC Cava are aged between 9 and 15 months and 78% of champagnes are aged between 12 and 36 months (data from the regulatory councils). Consequently, the majority of sparkling wines produced by the traditional method have percentages of polysaccharides and proteins from lees autolysis below 7%, and this value should even be lower in the youngest sparkling wines, especially those not produced by the traditional method.

As it was explained above, the bottles, in which the lees extracted from the sparkling wines were resuspended in a model wine solution, had been inserted with a pill for measuring dissolved oxygen by luminescence. The content of these bottles was saturated in oxygen and the oxygen concentration was monitored periodically for a year. **Figure 7** shows the oxygen consumption kinetics of the lees from sparkling wines from the first to the ninth year of aging time [22]. The oxygen consumption of the Control-A model wine solution (without adding lees) and the oxygen intake in Control-B (solutions without lees and oxygen) were very low and can be considered negligible (data not shown). In contrast, the oxygen consumption of all the samples containing lees increased over time, demonstrating that the lees can consume oxygen.

Moreover, this graph clearly shows that the lees of the first 3 years, especially those of the second year, consume much more oxygen than the lees of later years. It, therefore, seems clear that the ability of the lees of sparkling wines to consume oxygen increases between the first and second year and after tends to decrease throughout the aging period. The kinetic model proposed by Pascual et al. [38] was applied to these data to determine more precisely the total oxygen consumption capacity of the lees of these sparkling wines. **Figure 8** shows that the lees from the second year are capable of consuming nearly the double oxygen than those of the first or third year. Subsequently, the annual oxygen consumption decreases drastically in the older lees.

The higher oxygen consumption of the lees of the second year could be related to the described progress of the autolysis process which, according to some authors, starts slightly after 4 months and is more intense during the second year [7, 32, 39, 40]. It should also be noted that the maximal oxygen consumption-ability of the lees of the second-year match with the maximal polysaccharide and protein release and with the maximal levels of the foaming parameters [15]. All of these data seem to indicate that autolysis is at its peak during the second year of aging.

In any case, it seems that the oxygen consumption by the lees decreases drastically after 3 years of aging whereas the entrance of oxygen inside the sparkling wine

#### **Figure 7.**

*Oxygen consumption by lees extracted from sparkling wines of different aging times. Adapted from Pons-Mercadé et al. [22].*

*New Insights about the Influence of Yeasts Autolysis on Sparkling Wines Composition and Quality DOI: http://dx.doi.org/10.5772/intechopen.101314*

**Figure 8.**

*Total oxygen consumed in 1 year by the lees extracted from sparkling wines of different aging times. Adapted from Pons-Mercadé et al. [22].*

through the crown cap seems to be constant [41]. As long as the lees' oxygen consumption ability is greater than the oxygen permeation, the sparkling wine will be protected against oxidation. However, we can wonder what would happen when the lees stop consuming enough oxygen? When this happens, oxygen will be consumed by other wine components, especially by phenolic compounds, which will cause browning and the appearance of hydrogen peroxide that will oxidize other wine compounds in the absence of free sulfur dioxide, especially aroma compounds. Oxidation will be greater or lesser depending on the composition of the sparkling wine, which is largely dependent on the vintage and the production process.

**Figure 9** try to illustrate this complex balance showing the accumulated oxygen consumption by the lees in comparison with the oxygen intake across the crown cap considering the minimal value of oxygen permeability reported by Valade et al. [41]. The comparison of the two curves is just a theoretical approximation, but even so, it provides very interesting information.

According to this approach, the oxygen permeability across the crown cap remains below the accumulated oxygen consumed by the lees during the first 3 years of aging time and exceeds it at roughly three and a half years. More exactly the interception point is at 3 years and 7 months. This data indicates that after this aging time, the oxygen consumed by the lees would not be high enough to compensate for the oxygen entrance which would probably lead to wine oxidation. It should be taken into account that this calculation was done considering the minimal value of permeability reported for crown caps and that any increase in this permeability

#### **Figure 9.**

*Accumulation of oxygen consumed by the lees in comparison with the oxygen permeability of the crown cap. Adapted from Pons-Mercadé et al. [22].*

would therefore entail an earlier point of intersection in time. For instance, with a 20% higher permeability the intersection would take place just after 2 years of aging. As aforementioned, this is only a theoretical approach based on our results but it is very useful to illustrate what happens during sparkling wine aging.

All these sparkling wines were tasted by a trained panel and the main results are synthetized in **Figure 10**. The panel was asked to blindly classify sparkling wines based on their age. The panel successfully appreciated the chronological order of these sparkling wines since established four statistically significant groups depending on their sensory perception of their aging time: Group A, which the panel considered the youngest (first year of aging); Group B (second and third year of aging); Group C (fourth to sixth years of aging) and Group D (seventh to ninth years of aging).

All panelists considered the five youngest vintages of sparkling wines as "acceptable" for consumption under their qualitative sensory criterion. However, some of them considered that after this aging time the sparkling wines were "unacceptable". These data indicate that after 5 years the sparkling wines began to be affected by excessive ageing. It should be pointed out that these sensory data match well with the previous considerations about the balance between the oxygen consumption by the lees and the oxygen permeability across the crown cap. According to these results, the oxygen consumed by the lees started to be not enough to compensate for oxygen intake through the crown cap after 3 years and 7 months of ageing. After this time, the sparkling wine does not have enough defense against oxidation. Under these conditions, its sensory quality may begin to deteriorate, though the effects of this oxidation will also depend on its chemical composition and storage conditions. In the present study, sensory deterioration seems to begin after the 5th year of aging.

Finally, some photographs of the yeasts were taken using a scanning electron microscope (SEM) [15] to visualize the yeast autolysis process in the sparkling wines aged by up to 9 years (**Figure 11**). These pictures show how the structures of the yeast cells are progressively degraded, folded and deflated. In the first image, which shows the yeast of the starter culture used for the second fermentation of the last vintage (2016), the yeast cell seems very healthy since it is elongated, ovoid and turgid without any wrinkle or folds. Several bud scars can even be identified.

**Figure 10.**

*Sensory analysis of the sparkling wines of the nine consecutive vintages. Adapted from Pons-Mercadé et al. [15].*

*New Insights about the Influence of Yeasts Autolysis on Sparkling Wines Composition and Quality DOI: http://dx.doi.org/10.5772/intechopen.101314*

**Figure 11.**

*Monitoring yeast autolysis overtime using scanning electron microscopy. Adapted from Pons-Mercadé et al. [15].*

The second image shows what happens after the second fermentation (3 months later): the yeast cell has lost some turgor and is beginning to display wrinkles and folds. Two-years later, in the third year of ageing, the yeast cell is even more degraded and wrinkled and begins to deflate. At the fifth year of ageing, the yeast cell is completely flattened at the edges and retains only a little turgor in the middle, which is full of wrinkles and folds. In the seventh year of ageing, the yeast cell is even more degraded and deflated and the center of the cell has crumbled, wrinkled and flattened. Finally, in the ninth year, the yeast cell has completely collapsed and some of its structures are broken.
